Interphotoreceptor retinoid-Binding protein (IRBP) was shown by enzyme-linked immunosorbent assay (ELISA) to be absent from monkey liver, testes, lens and cornea. IRBP was present in cerebral cortex, pineal, vitreous and aqueous humor. These results were confirmed by Western Blot. IRBP was localized to rod photoreceptor cells in monkey retina by means of immunoelectronmicroscopy. Rats immunized with purified IRBP developed intraocular inflammatory changes and pineal gland inflammatory changes. Incubation of Y-79 retinoblastoma cells grown in monolayer tissue culture with radiolabeled precursors showed IRBP to be synthesized and secreted by these cells. IRBP synthesis was markedly increased by butyrate treatment. Purified monkey IRBP was found to contain 6.64 mol. of endogenous fatty acids per mol. protein. 60% of the total fatty acid was physically attached; the remaining 40% was covalently bound. Monkey, human and bovine IRBP were compared by analytical peptide mapping which showed monkey and human IRBP to be very similar and distinct from bovine IRBP. Tryptic peptides from bovine IRBP were isolated by reversed-phased high performance liquid chromatography (HPLC) and sequenced. One such peptide was used to confirm the identity of an IRBP clone.